DESCRIPTION: Respiratory and photosynthetic electron transport chains share common design features for separating charge across membranes, using the free energy of oxidation-reduction reactions. Across diverse phyla and even kingdoms, a small number of highly conserved membrane protein complexes are primarily responsible for these conversions. Among these, the cytochrome bc1 complex plays a central role in most systems. In spite of a broad knowledge-base on the functioning of the bcl complex, our understanding of the mechanism is very crude, and even gross features of its internal organization are subjects of controversy. The area of greatest ignorance is the nature of the charge separating reactions leading to electric potential generation ("electrogenesis"). In addition to electron transfer processes, which have been partially characterized by much prior work, these include proton transfer events that are almost completely obscure. This proposal focuses on the "electrogenic" steps of bc1 turnover, utilizing the kinetic advantages of light activation of the complex from Rhodobacter sphaeroides. The proposed research combines a direct electric potential measurement with optical spectroscopy, and the selective use of mutants, to address both electrogenic electron transfers between cofactors and the potentially electrogenic protonation steps of the quinone reactions in the bc1 complex. The specific aims include: (i) localization of the electrogenic steps and determination of the magnitudes of contributions from the high potential chain (QO-FeSR-c1-c2), bH-Qi electron transfer, and proton uptake to the Qi site; (ii) identification of the charge carrying species - whether electrons or protons, and (iii) characterization of the protolytic reactions resulting in proton uptake and proton release. To this end, protocols are devised for specific isolation of many steps in the turnover of the bcl complex, for study by electrometric and spectroscopic methods. These include: (i) Preparation of the system - reaction centers (RCs) and bcl - in defined states by changing redox potential, pH and ionic strength. (ii) Separation of the RC and bc1 reactions on the basis of two-flash experiment (distinguishing donor and acceptor sides of the bcl complex). (iii) Separation of reactions by removing electron-transport components (cytochrome c2, QB and other quinones) by traditional biochemical (extraction) methods; (iv) Isolation of different reactions by specific inhibitors of the bcl complex and RC. (v) Deconvolution of spectral, kinetic and thermodynamic parameters. (vi) Use of selected mutants to limit or eliminate specific reactions.